We propose to apply the aproaches of the organic chemist as well as the methods of the enzymologist to probe the nature of the active site of muscle creatine kinase, an enzyme which uses creatine, phosphocreatine, ADP and ATP as substrates. Creatine kinase thus serves a dual role as an objective for screening creatine, phosphocreatine and nucleoside triphosphate analogs of potentially great interest in other biological systems. Our general approach involves probing the enzyme's active site with synthetic substate analogs both to see which functional groups of the substrate are most vital to activity and to see what additional groups may be added without severe detrimental effect on activity. Various physical methods, including kinetic studies and nuclear magnetic resonance studies, are proposed in this probing process. The results of these specificity studies will be used in the design of affinity labels which are analogs of the normal substrates. Moreover, comparisons will be made between the substrate specificities of creatine kinase isozymes from different muscle sources such as skeletal muscle and heart tissues. It should be possible to design methods of detecting one isozyme in the presence of others based on these specificity differences.